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IL-33 released by alum is responsible for early cytokine production and has adjuvant properties.

Rose WA, Okragly AJ, Patel CN, Benschop RJ - Sci Rep (2015)

Bottom Line: Alum can induce the release of endogenous danger signals via cellular necrosis which elicits inflammation-associated cytokines resulting in humoral immunity.Furthermore, IL-33 itself functions as an adjuvant that, while only inducing a marginal primary response, facilitates a robust secondary response comparable to that observed with alum.Our results provide novel insights into the mechanism of action behind alum-induced cytokine responses and show that IL-33 is sufficient to provide a robust secondary antibody response independently of alum.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, 46285.

ABSTRACT
Human vaccines have used aluminium-based adjuvants (alum) for >80 years despite incomplete understanding of how alum enhances the immune response. Alum can induce the release of endogenous danger signals via cellular necrosis which elicits inflammation-associated cytokines resulting in humoral immunity. IL-33 is proposed to be one such danger signal that is released from necrotic cells. Therefore, we investigated whether there is a role for IL-33 in the adjuvant activity of alum. We show that alum-induced cellular necrosis results in elevated levels of IL-33 following injection in vivo. Alum and IL-33 induce similar increases in IL-5, KC, MCP-1, MIP-1α and MIP-1β; many of which are dependent on IL-33 as shown in IL-33 knockout mice or by using an IL-33-neutralizing recombinant ST2 receptor. Furthermore, IL-33 itself functions as an adjuvant that, while only inducing a marginal primary response, facilitates a robust secondary response comparable to that observed with alum. However, IL-33 is not absolutely required for alum-induced antibody responses since alum mediates similar humoral responses in IL-33 knockout and wild-type mice. Our results provide novel insights into the mechanism of action behind alum-induced cytokine responses and show that IL-33 is sufficient to provide a robust secondary antibody response independently of alum.

No MeSH data available.


Related in: MedlinePlus

Alum induces release of IL-33 via cellular necrosis.C57BL/6 mice (n = 5–8 mice/group) were injected i.p. with PBS or alum mixed with PBS at 1:2 ratio and peritoneal lavages were collected thirty minutes later. Peritoneal lavages were analyzed for IL-33 levels via ELISA (a) and percentage of necrotic cells using ViaCount solution which contains two proprietary DNA binding dyes to differentiate between viable, apoptotic, and dead cells (b). Data are combined from two independent experiments. **p < 0.01 PBS compared to alum injected groups (Two-tailed unpaired Student t-test). (c) Splenocytes isolated from naïve C57BL/6 mice were cultured with PBS (24 h) or alum mixed with PBS at a 1:2 ratio (n = 3 wells/group) for the indicated times then cellular necrosis was quantified as in (b). Data are representative of three independent experiments. ***p < 0.01 PBS compared to alum treated groups (One-way ANOVA with Dunnett’s test).
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f1: Alum induces release of IL-33 via cellular necrosis.C57BL/6 mice (n = 5–8 mice/group) were injected i.p. with PBS or alum mixed with PBS at 1:2 ratio and peritoneal lavages were collected thirty minutes later. Peritoneal lavages were analyzed for IL-33 levels via ELISA (a) and percentage of necrotic cells using ViaCount solution which contains two proprietary DNA binding dyes to differentiate between viable, apoptotic, and dead cells (b). Data are combined from two independent experiments. **p < 0.01 PBS compared to alum injected groups (Two-tailed unpaired Student t-test). (c) Splenocytes isolated from naïve C57BL/6 mice were cultured with PBS (24 h) or alum mixed with PBS at a 1:2 ratio (n = 3 wells/group) for the indicated times then cellular necrosis was quantified as in (b). Data are representative of three independent experiments. ***p < 0.01 PBS compared to alum treated groups (One-way ANOVA with Dunnett’s test).

Mentions: It has been reported that alum induces cellular necrosis and release of DNA following intraperitoneal (i.p.) injection19, and IL-33 is proposed to be released from necrotic cells as well222728. To show a potential direct link between alum-induced cellular necrosis and release of IL-33, we quantified IL-33 in the peritoneal cavity thirty minutes after i.p. injection of alum in mice. IL-33 levels were significantly increased in alum injected wild-type (WT) mice compared to PBS (Fig. 1a). IL-33 was not detected in IL-33 knockout (KO) mice injected with alum or PBS (data not shown). The percentage of necrotic cells isolated from the peritoneal cavity was significantly increased in the alum injected WT mice compared to PBS treated mice (Fig. 1b). To confirm these results and more directly quantify alum-induced cellular necrosis, splenocytes were isolated from naïve WT mice and cultured with alum or PBS for selected times. As shown in Fig. 1c, alum induced a significant increase in cellular necrosis over time. IL-33 levels from in vitro cultures were below the limit of detection for the IL-33 ELISA (data not shown). Thus, injection of alum into the peritoneal cavity resulted in rapid cellular necrosis and release of IL-33.


IL-33 released by alum is responsible for early cytokine production and has adjuvant properties.

Rose WA, Okragly AJ, Patel CN, Benschop RJ - Sci Rep (2015)

Alum induces release of IL-33 via cellular necrosis.C57BL/6 mice (n = 5–8 mice/group) were injected i.p. with PBS or alum mixed with PBS at 1:2 ratio and peritoneal lavages were collected thirty minutes later. Peritoneal lavages were analyzed for IL-33 levels via ELISA (a) and percentage of necrotic cells using ViaCount solution which contains two proprietary DNA binding dyes to differentiate between viable, apoptotic, and dead cells (b). Data are combined from two independent experiments. **p < 0.01 PBS compared to alum injected groups (Two-tailed unpaired Student t-test). (c) Splenocytes isolated from naïve C57BL/6 mice were cultured with PBS (24 h) or alum mixed with PBS at a 1:2 ratio (n = 3 wells/group) for the indicated times then cellular necrosis was quantified as in (b). Data are representative of three independent experiments. ***p < 0.01 PBS compared to alum treated groups (One-way ANOVA with Dunnett’s test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4536651&req=5

f1: Alum induces release of IL-33 via cellular necrosis.C57BL/6 mice (n = 5–8 mice/group) were injected i.p. with PBS or alum mixed with PBS at 1:2 ratio and peritoneal lavages were collected thirty minutes later. Peritoneal lavages were analyzed for IL-33 levels via ELISA (a) and percentage of necrotic cells using ViaCount solution which contains two proprietary DNA binding dyes to differentiate between viable, apoptotic, and dead cells (b). Data are combined from two independent experiments. **p < 0.01 PBS compared to alum injected groups (Two-tailed unpaired Student t-test). (c) Splenocytes isolated from naïve C57BL/6 mice were cultured with PBS (24 h) or alum mixed with PBS at a 1:2 ratio (n = 3 wells/group) for the indicated times then cellular necrosis was quantified as in (b). Data are representative of three independent experiments. ***p < 0.01 PBS compared to alum treated groups (One-way ANOVA with Dunnett’s test).
Mentions: It has been reported that alum induces cellular necrosis and release of DNA following intraperitoneal (i.p.) injection19, and IL-33 is proposed to be released from necrotic cells as well222728. To show a potential direct link between alum-induced cellular necrosis and release of IL-33, we quantified IL-33 in the peritoneal cavity thirty minutes after i.p. injection of alum in mice. IL-33 levels were significantly increased in alum injected wild-type (WT) mice compared to PBS (Fig. 1a). IL-33 was not detected in IL-33 knockout (KO) mice injected with alum or PBS (data not shown). The percentage of necrotic cells isolated from the peritoneal cavity was significantly increased in the alum injected WT mice compared to PBS treated mice (Fig. 1b). To confirm these results and more directly quantify alum-induced cellular necrosis, splenocytes were isolated from naïve WT mice and cultured with alum or PBS for selected times. As shown in Fig. 1c, alum induced a significant increase in cellular necrosis over time. IL-33 levels from in vitro cultures were below the limit of detection for the IL-33 ELISA (data not shown). Thus, injection of alum into the peritoneal cavity resulted in rapid cellular necrosis and release of IL-33.

Bottom Line: Alum can induce the release of endogenous danger signals via cellular necrosis which elicits inflammation-associated cytokines resulting in humoral immunity.Furthermore, IL-33 itself functions as an adjuvant that, while only inducing a marginal primary response, facilitates a robust secondary response comparable to that observed with alum.Our results provide novel insights into the mechanism of action behind alum-induced cytokine responses and show that IL-33 is sufficient to provide a robust secondary antibody response independently of alum.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, 46285.

ABSTRACT
Human vaccines have used aluminium-based adjuvants (alum) for >80 years despite incomplete understanding of how alum enhances the immune response. Alum can induce the release of endogenous danger signals via cellular necrosis which elicits inflammation-associated cytokines resulting in humoral immunity. IL-33 is proposed to be one such danger signal that is released from necrotic cells. Therefore, we investigated whether there is a role for IL-33 in the adjuvant activity of alum. We show that alum-induced cellular necrosis results in elevated levels of IL-33 following injection in vivo. Alum and IL-33 induce similar increases in IL-5, KC, MCP-1, MIP-1α and MIP-1β; many of which are dependent on IL-33 as shown in IL-33 knockout mice or by using an IL-33-neutralizing recombinant ST2 receptor. Furthermore, IL-33 itself functions as an adjuvant that, while only inducing a marginal primary response, facilitates a robust secondary response comparable to that observed with alum. However, IL-33 is not absolutely required for alum-induced antibody responses since alum mediates similar humoral responses in IL-33 knockout and wild-type mice. Our results provide novel insights into the mechanism of action behind alum-induced cytokine responses and show that IL-33 is sufficient to provide a robust secondary antibody response independently of alum.

No MeSH data available.


Related in: MedlinePlus